PHYTOCHEMISTRY Phytochemistry 68 (2007) 668672 www.elsevier.

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Constituents of the roots of Melochia chamaedrys

Dias G.C.D., Gressler V., Hoenzel S.C.S.M., Silva U.F., Dalcol I.I., Morel A.F.
mica, NPPN, Universidade Federal de Santa Maria, 97045-100 Santa Maria, RS, Brazil Departamento de Qu Received 12 January 2006; received in revised form 11 September 2006 Available online 13 December 2006

Abstract The cyclic peptide alkaloid, chamaedrine, was isolated from the roots of Melochia chamaedris (Sterculiaceae), along with four known cyclic Peptide alkaloids (adouetine X, frangulaline, scutianine B and scutianine C), and waltherione A, parasorbic acid, propacine, and ()-epicatequine. Their structures were elucidated on the basis of spectroscopic analysis, especially by 2D NMR (1H1H-COSY, NOESY, HMQC, HMBC). 2006 Elsevier Ltd. All rights reserved.
Keywords: Melochia chamaedrys; Sterculiaceae; Cyclic peptide alkaloid; Chamaedrine

1. Introduction The genus Melochia (Sterculiaceae), comprising nearly 65 species, is widespread in the tropics (Goldberg, 1967). Of these, Melochia corchorifolia Linn (Bhakuni et al., 1987, 1991) and Melochia tomentosa Linn (Goldberg, 1967; Kapadia et al., 1975, 1978, 1980, 1993; Shukla et al., 1976) are the most widely studied. Plants of this genus are used for relief of throat inammation (Morton, 1974), as tumorogenic agents (OGara et al., 1974), and as a cure for abdominal swelling, dysentery and water snakebite (Chopra et al., 1956). Plants belonging to this family are recognized to be rich in alkaloids, particularly the cyclopeptide alkaloids, quinolinone and isatin alkaloids (Goldberg, 1967; Shukla et al., 1976; Bhakuni et al., 1987, 1991; Kapadia et al., 1975, 1978, 1993).

Corresponding author. Tel.: +55 55 220 8205; fax: +55 55 220 8031. E-mail address: afmorel@base.ufsm.br (A.F. Morel).

Melochia chamaedris A. St.Hil, locally called douradinha, is a plant that grows in South America (Shouther Brazil, Argentine and Uruguay). In the Rio Grande do Sul (Brazil), M. chamaedris is used by the local population for the treatment of various diseases such as cancer and as an anti-hypertensive agent. However, nothing is known to date about its chemical constituents. In the course of our chemical studies on the alkaloidal components of South Brazilian medicinal plants, we have previously reported the isolation of cyclopeptide alkaloids (Morel et al., 1999a,b) and one quinolone (Hoelzel et al., 2005) from the bark of Waltheria douradinha, another genus of the Sterculiaceae family. In a continuation of this work, we now report the isolation and structural elucidation of chamaedrine (1), a cyclic peptide alkaloid from the roots of M. chamaedris. In addition, four known cyclic peptide alkaloids, adouetine X (2), frangufoline (3), scutianine B (4) and scutianine C (5), one quinolinone alkaloid, waltherione A (6), one lactone, parasorbic acid (7), one coumarinolignoid, propacine (8) and one avonoid, ()-epi-catechin (9) were also isolated.

0031-9422/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.11.004

G.C.D. Dias et al. / Phytochemistry 68 (2007) 668672

669

R' O O HN O R' N(CH3)2

5 2

R''

O NH
3

HN R' '

O O
O O

N H 6
7

O O O O
HO O

OH OH

O O
OH 9

OH

HO 8

2. Results and discussion Chamaedrine (1) was obtained as a white powder. Its molecular formula was determined as C36H41N5O4 by HRESIME ([M+H]+ = 608.32308 Da; [MH] = 606.30996 Da), in combination with analysis of the 13C NMR spectrum that showed 36 carbon resonances, including 21 methine, two methylenes, four methyls, and nine non-hydrogenated carbons, three of which had carbonyl groups. In the EIMS, the base peak appeared at m/z 148, corresponding to C10H14N, suggesting the presence of an N,N-dimethyl phenylalanine unit, which was conrmed by analysis of the 1H and 13C NMR spectroscopic data, and by enantioselective GC analyses. The 1H NMR spectrum (400 MHz, CDCl3,) of 1 showed two sets of doublets at d 0.87 (J = 6.7 Hz) and 1.15 (J = 6.7 Hz), which were assigned to the C-18 and C-19 methyl group hydrogens, respectively. The C-3 and C-4 methine hydrogens appeared at d 4.84 (dd, J = 8.0; 2.0 Hz) and 4.33 (dd, J = 10.5; 8.0 Hz), respectively. In the 1H1H-COSY spectrum, the two doublets of 18-Me and 19-Me have a cross-peak with the signal at d 1.85 (1H, m), which corresponds to H-17. In turn, the H-17 resonance shows a cross-peak with H-3 and this with H-4. H-4 also exhibits another cross-peak with H-20 (NH) at d 7.38 (d, J = 10.5 Hz). This spin system conrms b-hydroxyleucine as the hydroxylated amino acid of the

macrocycle ring. The vicinal coupling constant of ca. 8.0 Hz of the methine hydrogens (H-3/H-4) indicates an erythro conguration for this residue (Sierra et al., 1972). In addition, the NOESY spectrum of 1 (see Fig. 1) exhibits a NOE cross-peak between H-3 and H-14, suggesting that H-3 is located in the b-position. However, H-3 shows no cross-peak with H-4 indicating that they are in the antiposition. This evidence suggests that the b-hydroxyleucine moiety in 1 has a L-erythro conguration. In addition, from the 13C NMR spectra, the C-3 resonance observed at d 81.9 conrms that the b-hydroxyleucine moiety possesses a L-erythro (3S*, 4S*) conguration. The side-chain amino acid N,N-dimethyl phenylalanine was characterized by the occurrence of a double doublet at d 2.60 (dd, J = 4.5; 8.0 Hz), which was assigned to the H-22 methine hydrogen. It showed cross-peaks with H2-23 at d 2.70 (dd, J = 4.5; 14.0 Hz) and 2.81 (dd, J = 8.0; 14.0 Hz). The absolute stereochemistry of the N,N-dimethyl phenylalanine side-chain was determined as (S) by chiral phase gas chromatography using 3-pentyl-2,6-dimethyl-b-cyclodextrin (3-Pe-2,6-Me-b-CD) (Ko nig et al., 1990) as stationary phase. Tryptophan, which is the a-amino acid of the macrocycle, was identied by the cross-peaks between H2-30, H-7 and NH-6. The H2-30 at d 2.90 (dd, J = 8.0; 15.0 Hz) and 2.83 (dd, J = 4.0; 15.0 Hz) exhibits a cross-peak with H-7 at d 4.32 (m) and H-7 shows another cross-peak with H-6 (NH) at d 5.75 (d, J = 8.0). In the NOESY spectrum, the H-4 signal exhibits a cross-peak with H-6 (NH), but does not show a cross-peak with H-7. This suggests that the a-amino acid tryptophan has an L (S*) conguration. The 13C NMR spectrum (100.6 MHZ, CDCl3) of 1 indicated the presence of 36 carbons. DEPT, HMQC and HMBC experiments showed the presence of two methylene, four methyls, 21 methine and nine non-hydrogenated carbons. The signals at dC 172.6, 171.5 and 167.2 permitted the assignment of three carbonyl groups in 1. In the HMBC spectrum, cross-peaks between dH 2.60 (H-22)
15 14 18 17 19 16 13 12 11 10 8

H O
3

H O H N O
5 6

O
7

HN20
21 22 23 24 25

NH H
39

34

35 36

30 33

N(CH3)2
28,29

38 32

37

N 31 H

26 27

Fig. 1. Cyclopeptide correlations.

alkaloid

and

important

H1H-NOESY

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G.C.D. Dias et al. / Phytochemistry 68 (2007) 668672

and dH 2.70/2.81 (H2-23) to dC 172.6 permitted the assignment of a carbonyl group at C-21, while cross-peaks between dH 4.84 (H-3) and dH 4.33 (H-4) to dC 171.5, and dH 4.32 (H-7) to dC 167.2, were indicative of carbonyl groups at C-5 and C-8, respectively. The 1H and 13C NMR spectra of 1, along with the coupling constants of the hydrogens and the most important connectivities observed in the HMBC spectrum, are summarized in Table 1. A number of known compounds were also isolated from this source for the rst time. Alkaloids 26 were identied by comparison with authentic samples of adouetine X (2), frangufoline (3), scutianine B (4), scutianine C (5), and waltherione A (6), whereas compounds 79 were identied by comparing their spectroscopic data (1H NMR and 13C NMR) with the literature (Cardellina and Meinwald, et al., 1991). 1980; Zoghbi et al., 1981; Balde Our results on M. chamaedris conrm previous ndings that members of the genus Melochia contain cyclic peptide alkaloids and quinolinone alkaloids (Goldberg, 1967; Shukla et al., 1976; Bhakuni et al., 1987, 1991; Kapadia et al., 1975, 1978, 1993). The nding of parasorbic acid (7) and propacine (8) in M. chamaedris was unexpected since such compounds have not been recorded from the family Sterculiaceae before.

3. Experimental 3.1. General General experimental procedures: Melting points were determined with an MQAPF-301 apparatus and are uncorrected. Optical rotations were taken on a Perkin Elmer 341 digital polarimeter. High-resolution ESI mass spectra were recorded on a Bruker Bio Apex 70 eV FTICR (Bruker Daldonis, USA) instrument. Low resolution MS were recorded at an ionization potential of 70 eV. 1H and 13C NMR spectra were recorded at 400.1/100.6 MHz, on a Bruker DPX-400 spectrometer using CDCl3 as solvent and TMS as internal standard. Thin layer chromatography was performed on a pre-coated TLC plates (Merk, silica 60 F-254), by spraying with Dragendor reagent, and by spraying with 10% H2SO4 in EtOH, followed by heating. 3.2. Plant material The plant material (roots) was collected in February May 2001 in the state Rio Grande do Sul, Brazil. Prof. Thais S.C. Dorow identied the plant and a voucher specimen (SMDB 9262) is deposited at the herbarium of the University of Santa Maria.

Table 1 1 H and 13C NMR spectroscopic data for chamaedrine (1) (in CDCl3, 400/100.6 MHz)a H/C d 1H (J, Hz) d
13

C (ppm)

HMBC correlations
2

JCH

JCH

01 03 04 05 06 07 08 09 10 11 12 1316 17 18 19 20 21 22 23 24 2527 2829 30 31 3437 38 39

a

4.84 dd (2.0, 8.0) 4.33 dd (8.0, 10.5) 5.75 d (8.0) 4.32 m 6.40 d (7.5) 5.75 (signal overlapping) 5.76 (signal overlapping) 7.07.5 1.85 m 0.87 d (6.7) 1.15 d (6.7) 7.38 d (10.5 ) 2.60 dd (4.5, 8.0) 2.70 dd (4.5, 14.0)/2.81 dd (8.0, 14.0 )

156.0 81.8 54.9 171.5 54.0 167.2 131.4 126.9 131.6 120.0129.4 14.0 20.3 172.6 69.3 141.0 119.0130.0 41.5 39.2 136.0 126.6

C-17 C-5 C-8 C-11 C-10, C-12 C-17 C-17 C-21, C-23 C-22 C-7

C-1, C-5 C-5 C-12 C-1

C-24

2.12 m 2.83 dd (4.5, 15.0) /2.90 dd (8.0, 15.0) 9.55 (br s)

Assignments were obtained by analyses 2D 1H1H-COSY, NOESY, DEPT and 2D 1H13C COSY (HMQC, HMBC) experiments.

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3.3. Extraction and isolation Air-dried roots of M. chamaedris (780 g) were pulverized and extracted with MeOH (3 l 5, 5 days each time), at room temperature. Removal of the solvent from the combined extracts was performed under reduced pressure in a rotary evaporator to obtain a dark crude extract (200 g). This extract was next suspended in H2O (0.3 l) and successively partitioned with n-hexane (HF, yield 10 g), CHCl3 (CF, yield 4.5 g), EtOAc (AcF, yield 11 g) and n-BuOH (BuF, 28 g). The CHCl3 residue (CF) containing six Dragendor-positive spots in TLC, was subjected to silica gel CC eluted with a gradient of CHCl3 containing increasing amounts of MeOH (up to 10% to give 15 fractions). Fraction 2 (CHCl3:MeOH, 99.5:0.5) consisting of one compound, was evaporated to give 7 (210 mg). Fraction 3 (CHCl3:MeOH, 99.5:0.5), consisting of two compounds, was evaporated (55 mg) and submitted to preparative TLC (CHCl3:MeOH, 99.5:0.5, two elutions) to yield 7 (10 mg) and 8 (35 mg). Fraction 5 (CHCl3:MeOH, 99:1), containing two Dragendor-positive spots by TLC, was evaporated (79 mg) and submitted to preparative TLC (CHCl3:MeOH, 99:1, two elutions) to yield 1 (9 mg) and 2 (25 mg). Fractions 6 and 7 (CHCl3:MeOH, 98:2), containing three Dragendor-positive spots in TLC, were combined (160 mg) and resubmitted to silica gel CC (19 g) with CHCl3 containing increasing amounts of MeOH (up to 4%) to yield 3 (20 mg), 4 (75 mg) and 5 (45 mg). Fraction 8 (CHCl3:MeOH, 98:2) consisting of two alkaloids, was evaporated (55 mg) and submitted to preparative TLC (CHCl3 MeOH, 98:2, two elutions) to yield 5 (20 mg) and 6 (25 mg). The EtOAc fraction (1 g) was applied to a silica gel column (80 g) and eluted with toluene containing increasing amounts of EtOAc (up to 50%) to give 12 fractions. Fractions 58 (toluene:EtOAc, 4:1) containing only one FeCl3-positive spots in TLC (120 mg) was submitted to preparative TLC (toluene:EtOAc, 2:1) to yield 9 (70 mg). 3.4. Chamaedrine (1) White powder; m.p. 222223 C, aD : 122.5 (c 0.12 CHCl3). IR (KBr): mmax 3437, 3272, 1640 cm. HRESIME ([M+H]+ at m/z 608. 32308 (C36H42N5O4, requires 608.32331). EIMS: m/z = 607 [M]+, 190, 148 (100%), 135, 130. For 1H and 13C NMR spectroscopic data (400 MHz, CDCl3 and 100 MHz, CDCl3), and HMBC, see Table 1. 3.5. Hydrolysis and amino acid derivatization Hydrolysis of chamaedrine (1) (3 mg) was performed in a sealed tube at 110 C with 6 N HCl for 24 h as previously described (Silva et al., 1996). The acidic solution was concentrated and the residue was used to identify the absolute stereochemistry of N,N-dimethyl phenylalanine. Acid-catalyzed esterication of the resulting amino acids (2 mg) was carried out by the addition of a 1.6 N anhydrous solution
25

of HCl gas in MeOH for 30 min at room temperature (Bayer and Konig, 1969). After removal of the reagents under N2, the sample was taken up in CH2Cl2 (200 ll) and triuoroacetic anhydride (50 ll). The mixture was kept at room temperature for 30 min and the excess reagent was removed under a dry N2 stream. 3.6. GC analysis of N,N-dimethyl phenylalanine The derivatized amino acid was analyzed by enantioselective capillary GC by employing modied cyclodextrin as chiral stationary phase and by coinjection with standard L- and D, L-amino acids (Bayer and Konig, 1969). N,Ndimethyl phenylalanine was obtained as previously described (Mcdermott and Benoiton, 1973).

Acknowledgements ncia e The authors thank SCT (Secretaria da Cie Tecnologia do Estado do Rio Grande do Sul) and CNPq co e Tec(Conselho Nacional de Desenvolvimento Cient gico) for nancial support. nolo

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